Human iPSC-liver organoid transplantation reduces fibrosis through immunomodulation.
Tomomi Tadokoro, Soichiro Murata, Mimoko Kato, Yasuharu Ueno, Tomonori Tsuchida, Ayumu Okumura, Yoshiki Kuse, Takahiro Konno, Yutaro Uchida, Yuriko Yamakawa, Marina Zushi, Megumi Yajima, Tatsuya Kobayashi, Shunsuke Hasegawa, Yumi Kawakatsu-Hatada, Yoshihito Hayashi, Shun Osakabe, Takuji Maeda, Kodai Kimura, Akihiro Mori, Maiko Tanaka, Yu Kamishibahara, Megumi Matsuo, Yun-Zhong Nie, Satoshi Okamoto, Takayoshi Oba, Naoki Tanimizu, Hideki Taniguchi
Science translational medicine, 16, 757, eadg0338, 2024年07月24日, ［国際誌］
英語, 研究論文（学術雑誌）, Donor organ shortages for transplantation remain a serious global concern, and alternative treatment is in high demand. Fetal cells and tissues have considerable therapeutic potential as, for example, organoid technology that uses human induced pluripotent stem cells (hiPSCs) to generate unlimited human fetal-like cells and tissues. We previously reported the in vivo vascularization of early fetal liver-like hiPSC-derived liver buds (LBs) and subsquent improved survival of recipient mice with subacute liver failure. Here, we show hiPSC-liver organoids (LOs) that recapitulate midgestational fetal liver promote de novo liver generation when grafted onto the surface of host livers in chemical fibrosis models, thereby recovering liver function. We found that fetal liver, a hematopoietic tissue, highly expressed macrophage-recruiting factors and antifibrotic M2 macrophage polarization factors compared with the adult liver, resulting in fibrosis reduction because of CD163+ M2-macrophage polarization. Next, we created midgestational fetal liver-like hiPSC-LOs by fusion of hiPSC-LBs to induce static cell-cell interactions and found that these contained complex structures such as hepatocytes, vasculature, and bile ducts after transplantation. This fusion allowed the generation of a large human tissue suitable for transplantation into immunodeficient rodent models of liver fibrosis. hiPSC-LOs showed superior liver function compared with hiPSC-LBs and improved survival and liver function upon transplantation. In addition, hiPSC-LO transplantation ameliorated chemically induced liver fibrosis, a symptom of liver cirrhosis that leads to organ dysfunction, through immunomodulatory effects, particularly on CD163+ phagocytic M2-macrophage polarization. Together, our results suggest hiPSC-LO transplantation as a promising therapeutic option for liver fibrosis.

Induced pluripotent stem cell-derived hematopoietic stem and progenitor cells induce mixed chimerism and donor-specific allograft tolerance.
Tomoki Murata, Naoki Hama, Tomoki Kamatani, Akihiro Mori, Ryo Otsuka, Haruka Wada, Ken-Ichiro Seino
American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 23, 9, 1331, 1344, 2023年09月, ［国際誌］
英語, 研究論文（学術雑誌）, In transplantation using allogeneic induced pluripotent stem cells (iPSCs), strategies focused on major histocompatibility complexes were adopted to avoid immune rejection. We showed that minor antigen mismatches are a risk factor for graft rejection, indicating that immune regulation remains one of the most important issues. In organ transplantation, it has been known that mixed chimerism using donor-derived hematopoietic stem/progenitor cells (HSPCs) can induce donor-specific tolerance. However, it is unclear whether iPSC-derived HSPCs (iHSPCs) can induce allograft tolerance. We showed that 2 hematopoietic transcription factors, Hoxb4 and Lhx2, can efficiently expand iHSPCs with a c-Kit+Sca-1+Lineage- phenotype, which possesses long-term hematopoietic repopulating potential. We also demonstrated that these iHSPCs can form hematopoietic chimeras in allogeneic recipients and induce allograft tolerance in murine skin and iPSC transplantation. With mechanistic analyses, both central and peripheral mechanisms were suggested. We demonstrated the basic concept of tolerance induction using iHSPCs in allogeneic iPSC-based transplantation.

Evaluation of immunosuppression protocols for MHC-matched allogeneic iPS cell-based transplantation using a mouse skin transplantation model.
Tomoki Kamatani, Ryo Otsuka, Tomoki Murata, Haruka Wada, Takeshi Takahashi, Akihiro Mori, Soichiro Murata, Hideki Taniguchi, Ken-Ichiro Seino
Inflammation and regeneration, 42, 1, 4, 4, 2022年02月02日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Off-the-shelf major histocompatibility complex (MHC)-matched iPS cells (iPSC) can potentially initiate host immune responses because of the existence of numerous minor antigens. To suppress allo-immune responses, combination of immunosuppressants is usually used, but its efficacy to the allogeneic iPSC-based transplantation has not been precisely evaluated. METHODS: Three transplantation models were used in this study; MHC-matched, minor antigen-mismatched mouse skin or iPSC-graft transplantation, and fully allogeneic human iPSC-derived liver organoid transplantation in immune-humanized mice. The recipients were treated with triple drugs combination (TDC; tacrolimus, methylprednisolone, and mycophenolate mofetil) or co-stimulatory molecule blockade (CB) therapy with some modifications. Graft survival as well as anti-donor T and B cell responses was analyzed. RESULTS: In the mouse skin transplantation model, immunological rejection caused by the minor antigen-mismatch ranged from mild to severe according to the donor-recipient combination. The TDC treatment could apparently control the mild skin graft rejection when combined with a transient T cell depletion, but unexpected anti-donor T or B cell response was observed. On the other hand, CB therapy, particularly when combined with rapamycin treatment, was capable of attenuating both mild and severe skin graft rejection and allowing them to survive long-term without any unfavorable anti-donor immune responses. The efficacy of the CB therapy was confirmed in both mouse and human iPSC-derived graft transplantation. CONCLUSIONS: The findings suggest that the CB-based treatment seems suitable to well manage the MHC-matched allogeneic iPSC-based transplantation. The TDC-based treatment may be also used to suppress the rejection, but screening of its severity prior to the transplantation seems to be needed.

Serous Membrane Detachment with Ultrasonic Homogenizer Improves Engraftment of Fetal Liver to Liver Surface in a Rat Model of Cirrhosis.
Yumi Kawakatsu-Hatada, Soichiro Murata, Akihiro Mori, Kodai Kimura, Hideki Taniguchi
International journal of molecular sciences, 22, 21, 2021年10月27日, ［国際誌］
英語, 研究論文（学術雑誌）, Liver transplantation is the most effective treatment for end-stage cirrhosis. However, due to serious donor shortages, new treatments to replace liver transplantation are sorely needed. Recent studies have focused on novel therapeutic methods using hepatocytes and induced pluripotent stem cells, we try hard to develop methods for transplanting these cells to the liver surface. In the present study, we evaluated several methods for their efficiency in the detachment of serous membrane covering the liver surface for transplantation to the liver surface. The liver surface of dipeptidyl peptidase IV (DPPIV)-deficient rats in a cirrhosis model was detached by various methods, and then fetal livers from DPPIV-positive rats were transplanted. We found that the engraftment rate and area as well as the liver function were improved in rats undergoing transplantation following serous membrane detachment with an ultrasonic homogenizer, which mimics the Cavitron Ultrasonic Surgical Aspirator® (CUSA), compared with no detachment. Furthermore, the bleeding amount was lower with the ultrasonic homogenizer method than with the needle and electric scalpel methods. These findings provide evidence that transplantation to the liver surface with serous membrane detachment using CUSA might contribute to the development of new treatments for cirrhosis using cells or tissues.

A Novel Orthotopic Liver Cancer Model for Creating a Human-like Tumor Microenvironment.
Rong Qiu, Soichiro Murata, Chao Cheng, Akihiro Mori, Yunzhong Nie, Satoshi Mikami, Shunsuke Hasegawa, Tomomi Tadokoro, Satoshi Okamoto, Hideki Taniguchi
Cancers, 13, 16, 2021年08月08日, ［国際誌］
英語, 研究論文（学術雑誌）, Hepatocellular carcinoma (HCC) is the most common form of liver cancer. This study aims to develop a new method to generate an HCC mouse model with a human tumor, and imitates the tumor microenvironment (TME) of clinical patients. Here, we have generated functional, three-dimensional sheet-like human HCC organoids in vitro, using luciferase-expressing Huh7 cells, human iPSC-derived endothelial cells (iPSC-EC), and human iPSC-derived mesenchymal cells (iPSC-MC). The HCC organoid, capped by ultra-purified alginate gel, was implanted into the disrupted liver using an ultrasonic homogenizer in the immune-deficient mouse, which improved the survival and engraftment rate. We successfully introduced different types of controllable TME into the model and studied the roles of TME in HCC tumor growth. The results showed the role of the iPSC-EC and iPSC-MC combination, especially the iPSC-MC, in promoting HCC growth. We also demonstrated that liver fibrosis could promote HCC tumor growth. However, it is not affected by non-alcoholic fatty liver disease. Furthermore, the implantation of HCC organoids to humanized mice demonstrated that the immune response is important in slowing down tumor growth at an early stage. In conclusion, we have created an HCC model that is useful for studying HCC development and developing new treatment options in the future.

Establishment of Human Leukocyte Antigen-Mismatched Immune Responses after Transplantation of Human Liver Bud in Humanized Mouse Models.
Akihiro Mori, Soichiro Murata, Nao Tashiro, Tomomi Tadokoro, Satoshi Okamoto, Ryo Otsuka, Haruka Wada, Tomoki Murata, Takeshi Takahashi, Ken-Ichiro Seino, Hideki Taniguchi
Cells, 10, 2, 2021年02月23日, ［国際誌］
英語, 研究論文（学術雑誌）, Humanized mouse models have contributed significantly to human immunology research. In transplant immunity, human immune cell responses to donor grafts have not been reproduced in a humanized animal model. To elicit human T-cell immune responses, we generated immune-compromised nonobese diabetic/Shi-scid, IL-2RγKO Jic (NOG) with a homozygous expression of human leukocyte antigen (HLA) class I heavy chain (NOG-HLA-A2Tg) mice. After the transplantation of HLA-A2 human hematopoietic stem cells into NOG-HLA-A2Tg, we succeeded in achieving alloimmune responses after the HLA-mismatched human-induced pluripotent stem cell (hiPSC)-derived liver-like tissue transplantation. This immune response was inhibited by administering tacrolimus. In this model, we reproduced allograft rejection after the human iPSC-derived liver-like tissue transplantation. Human tissue transplantation on the humanized mouse liver surface is a good model that can predict T-cell-mediated cellular rejection that may occur when organ transplantation is performed.

Investigation of Clinical Safety of Human iPS Cell-Derived Liver Organoid Transplantation to Infantile Patients in Porcine Model.
Tomonori Tsuchida, Soichiro Murata, Shunsuke Hasegawa, Satoshi Mikami, Shin Enosawa, Huai-Che Hsu, Akinari Fukuda, Satoshi Okamoto, Akihiro Mori, Megumi Matsuo, Yumi Kawakatsu, Hitomi Matsunari, Kazuaki Nakano, Hiroshi Nagashima, Hideki Taniguchi
Cell transplantation, 29, 963689720964384, 963689720964384, 2020年, ［国際誌］
英語, 研究論文（学術雑誌）, Transplantation of liver organoids has been investigated as a treatment alternative to liver transplantation for chronic liver disease. Transportal approach can be considered as a method of delivering organoids to the liver. It is important to set the allowable organoid amount and verify translocation by intraportal transplantation. We first examined the transplantation tolerance and translocation of porcine fetal liver-derived allogeneic organoids using piglets. Fetal liver-derived organoids generated from the Kusabira Orange-transduced pig were transplanted to the 10-day-old piglet liver through the left branch of the portal vein. All recipients survived without any observable adverse events. In contrast, both local and main portal pressures increased transiently during transplantation. In necropsy samples, Kusabira Orange-positive donor cells were detected primarily in the target lobe of the liver and partly in other areas, including the lungs and brain. As we confirmed the transplantation allowance by porcine fetal liver-derived organoids, we performed intraportal transplantation of human-induced pluripotent stem cell (iPSC)-derived liver organoid, which we plan to use in clinical trials, and portal pressure and translocation were investigated. Human iPSC-derived liver organoids were transplanted into the same 10-day-old piglet. Portal hypertension and translocation of human iPSC-derived liver organoids to the lungs were observed in one of two transplanted animals. Translocation occurred in the piglet in which patent ductus venosus (PDV) was observed. Therefore, a 28-day-old piglet capable of surgically ligating PDV was used, and after the PDV was ligated, human iPSC-derived liver organoids with the amount of which is scheduled in clinical trials were transplanted. This procedure inhibited the translocation of human iPSC-derived liver organoids to extrahepatic sites without no portal hypertension. In conclusion, human iPSC-derived liver organoids can be safely transplanted through the portal vein. Ligation of the ductus venosus prior to transplantation was effective in inhibiting extrahepatic translocation in newborns and infants.

The Regenerative Effect of Portal Vein Injection of Liver Organoids by Retrorsine/Partial Hepatectomy in Rats.
Tomonori Tsuchida, Soichiro Murata, Koichiro Matsuki, Akihiro Mori, Megumi Matsuo, Satoshi Mikami, Satoshi Okamoto, Yasuharu Ueno, Tomomi Tadokoro, Yun-Wen Zheng, Hideki Taniguchi
International journal of molecular sciences, 21, 1, 2019年12月26日, ［国際誌］
英語, 研究論文（学術雑誌）, In this study, we reveal that liver organoid transplantation through the portal vein is a safe and effective method for the treatment of chronic liver damage. The liver organoids significantly reconstituted the hepatocytes; hence, the liver was significantly enlarged in this group, compared to the monolayer cell transplantation group in the retrorsine/partial hepatectomy (RS/PH) model. In the liver organoid transplantation group, the bile ducts were located in the donor area and connected to the recipient bile ducts. Thus, the rate of bile reconstruction in the liver was significantly higher compared to that in the monolayer group. By transplanting liver organoids, we saw a level of 70% replacement of the damaged liver. Consequently, in the transplantation group, diminished ductular reaction and a decrease of placental glutathione S-transferase (GST-p) precancerous lesions were observed. After trans-portal injection, the human induced pluripotent stem cell (hiPSC)-derived liver organoids revealed no translocation outside the liver; in contrast, the monolayer cells had spread to the lungs. The hiPSC-derived liver organoids were attached to the liver in the immunodeficient RS/PH rats. This study clearly demonstrates that liver organoid transplantation through the portal vein is a safe and effective method for the treatment of chronic liver damage in rats.

iPS細胞が誘導する免疫寛容（TIPS）のメカニズム解明と応用
科学研究費助成事業
2024年06月28日 - 2027年03月31日
清野 研一郎, 森 淳祐
日本学術振興会, 挑戦的研究(開拓), 北海道大学, 24K21299